The enzymatic hydrolysis of 5-substituted hydantoins to N-carbamoyl-amino acids and the further reaction thereof to the corresponding enantiomerically-enriched amino acids is a standard method in organic chemistry (“Enzyme Catalysis in Organic Synthesis”, Eds.: Drauz, Waldmann, VCH, 1st and 2nd Ed.). The enantiodifferentiation can be carried out either at the stage of the hydantoin hydrolysis by means of hydantoinases or alternatively during the cleavage of the N-carbamoyl-amino acids by means of enantioselective carbamoylases. Because the enzymes each convert only one optical antipode of the corresponding compound, it is attempted to racemise the other in the mixture (in situ) in order to ensure the complete conversion of the hydantoin, which can readily be prepared racemically, into the corresponding enantiomerically enriched amino acid. The racemisation can proceed either at the stage of the hydantoins by means of chemical (base, acid, elevated temperature) or enzymatic processes or alternatively at the stage of the N-carbamoyl-amino acids by means of, for example, acetylamino acid racemases (DE10050124). By its nature, the latter variant is only successful if enantioselective carbamoylases are used. The following scheme illustrates this fact.

For aromatic substrates, the rate of the chemical racemisation of the hydantoins, as shown in Table 1, is sufficiently high to ensure high space-time yields for the preparation of amino acids by the hydantoinase process. For aliphatic hydantoins, such as isobutyl-, methyl- and isopropyl-hydantoin, however, the racemisation represents a considerable bottleneck in the synthesis of aliphatic amino acids.
TABLE 1Racemisation constants of hydantoins at 40° C.,pH 8.5 determined by initial rates according to a first-order reaction (−krac = ln([a]/[a0]) from: Hydrolysis andFormation of Hydantoins (Chpt. B 2.4). Syldatk, C. andPietzsch, M. In: Enzyme catalysis in organic synthesis(Eds.: K. Drauz & H. Waldmann), VCH, 1st and 2nd Ed.).5′-substituentkrac (h−1)t1/2 (h)Phenyl2.590.27Methylthioethyl0.125.82Isobutyl0.03221.42Methyl0.0233.98Isopropyl0.01255.90
This problem manifests itself, for example, in the preparation, described in EP759475, of enantiomerically enriched tert.-butylhydantoin by means of the hydantoinase process. In this case, the complete conversion of 32 mM tert.-butylhydantoin with 1.5 kU R-hydantoinase required 8 days at pH 8.5 and 4 days at pH 9.5. The low space-time yield is in fact caused by the only slow chemical racemisation of tert.-butylhydantoin (krac=0.009 h−1 at 50° C. and pH 8.5).
There are known from the prior art hydantoin racemases from microorganisms of the genus Pseudomonas, Microbacterium, Agrobacterium and Arthrobacter (lit.: JP04271784; EP1188826; Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation. Hils, M.; Muench, P.; Altenbuchner, J.; Syldatk, C.; Mattes, R. Applied Microbiology and Biotechnology (2001), 57(5-6), 680-688; A new razemase for 5-monosubstituted hydantoins. Pietzsch, Markus; Syldatk, Christoph; Wagner, Fritz. Ann. N. Y. Acad. Sci. (1992), 672 (Enzyme Engineering XI), 478-83. Lickefett, Holger; Krohn, Karsten; Koenig, Wilfried A.; Gehrcke, Barbel; Syldatk, Christoph. Tetrahedron: Asymmetry (1993), 4(6), 1129-35; Purification and characterization of the hydantoin razemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli. Watabe, Ken; Ishikawa, Takahiro; Mukohara, Yukuo; Nakamura, Hiroaki. J. Bacteriol. (1992), 174(24), 7989-95).
Of the hydantoin racemases from Arthrobacter aurescens DSM 3745, Pseudomonas sp. NS671 and Microbacterium liquefaciens, it is known that these enzymes racemise aliphatic hydantoins, such as, for example, isopropylhydantoin or isobutylhydantoin, only weakly. It is also known that the hydantoin racemases from Arthrobacter aurescens DSM 3747 preferentially convert aromatic hydantoins, such as indolylmethylhydantoin or benzylhydantoin, whereas aliphatic hydantoins, such as methylthioethylhydantoin, are converted comparatively weakly or, in the case of isopropylhydantoin, are not converted at all (A new razemase for 5-monosubstituted hydantoins. Pietzsch, Markus; Syldatk, Christoph; Wagner, Fritz. Ann. N. Y. Acad. Sci. (1992), 672 (Enzyme Engineering XI), 478-83.).
The low activity of hydantoin racemases therefore frequently limits the economic potential of this route.